Les auteurs remercient le Dr Le Trinh et le Dr Tobin pour fournir généreusement l&#39;α-caténine: ligne de poissons de citrine et Bill Jackman pour nous permettre de faire le tournage dans son laboratoire. Les auteurs reconnaissent les sources de financement Instituts nationaux de la santé (subventions 5P20RR016463, 8P20GM103423 et R15AI094406) et l&#39;USDA (Projet # ME0-H-1-00517-13). Ce manuscrit est publié en tant que principal Agriculture et foresterie Expérience publication station numéro 3371.

REMARQUE: Tous les protocoles et les expériences de soins de poisson zèbre ont été effectuées conformément aux directives du NIH sous institutionnel de protection des animaux et l&#39;utilisation Comité (IACUC) A2012-11-03 protocole. 1. poisson zèbre élevage pour quatre jours après la fécondation <ol><li> Recueillir AB poisson zèbre, ou d&#39;autres lignées transgéniques, dans le premier poste de fertilisation 3 heures, comme montré dans un autre 34 vidéo. </li><li> Incuber 120 oeufs dans un 15 cm boîtes de Pétri contenant 150 ml de milieu E3 (mM de NaCl 5; KCl mM 0,17; 0,33 mM de CaCl2; 0,33 mM de MgCl2, 2 mM d&#39;HEPES, pH 6,8) avec du bleu de méthylène (0,3 ug / ml final concentration) dans un incubateur à 33 ° avec la lumière 12/12 / photopériode sombre. </li><li> Remplacez le support après 6 h avec E3 + PTU (1-phényl-2-thiourée, 10 pg / ml de concentration finale) et retirer les oeufs morts. </li><li> Incuber pendant 4 jours à 33 ° C, l&#39;échange avec les médias frais E3 + PTU après deux jours. </li></ol> 2. Dansprojection Micro-aiguille Préparation <ol><li> Tirez borosilicate capillaire (OD 1,2 mm; ID 0,69 mm) dans un micro-aiguille à l&#39;aide d&#39;un extracteur de l&#39;aiguille avec les paramètres suivants (550 chaleur; 0 Pull; 130 Velocity; 110 Time). REMARQUE: Les paramètres varient d&#39;filament filament pour obtenir des micro-aiguilles de forme similaire (voir la figure 1) et devra être réajusté chaque fois qu&#39;un nouveau filament est installé. </li><li> Remplir une micro-aiguille avec 3 pi de 0,4 um filtré la solution saline de tampon phosphate (PBS; NaCl 137 mM; KCl 2,7 mM; 10 mM de Na 2 HPO 4, 1,8 mM de KH 2 PO 4; pH 7,4) en utilisant un microloader pipette 20 ul pointe. </li><li> Mettre en place la micro-aiguille sur un support de micropipette sur un micromanipulateur relié à un système d&#39;injection sous pression. Régler la pression d&#39;injection à 30 PSI et la durée d&#39;impulsion à 30 ms. </li><li> Utilisation d&#39;une boîte de Pétri avec de l&#39;eau distillée, abaisser la micro-aiguille jusqu&#39;à 1/5 de l&#39;aiguille est dans l&#39;eau. Clip de la micro-aiguille with pinces fines juste en dessous du niveau de l&#39;eau. Apportez la micro-aiguille hors de l&#39;eau et pousser la pédale à plusieurs reprises jusqu&#39;à ce qu&#39;un bolus apparaît. </li><li> Mesurer le diamètre du bol par l&#39;abaissement de la micro-aiguille sur un hémocytomètre en couches avec une goutte de type A de l&#39;huile d&#39;immersion. Réglez la durée d&#39;impulsion pour obtenir le volume souhaité (voir tableau 1). </li><li> Régler la contre-pression pour le bolus de rester stable dans l&#39;huile (abaisser la pression si le bolus augmente le volume et augmenter la contre-pression si le volume de bolus diminue). Enregistrer la durée d&#39;impulsion pour chaque micro-aiguille. </li><li> Retirez le PBS de la micro-aiguille en aspirant à l&#39;aide d&#39;une pipette de microloader et d&#39;expulser les restes de PBS en la replaçant sur l&#39;injecteur et appuyer sur l&#39;interrupteur d&#39;impulsion continue. Réservez chaque micro-aiguille. </li></ol> 3. Préparation Candida <ol><li> Streak Candida albicans transformées avec codon optimisé dTomato, GFP, BFP, EOS ou en Extrême-Rouge à partir d&#39;un stock congelé (-80 ° C stock de 25% de glycérol) en utilisant une cheville en bois stérile sur un dextrose levure Peptine (YPD) plaque de gélose (10 g d&#39;extrait / L de levure, 20 g / L peptone, 20 g / L de dextrose , / L agar 20 g; autoclave et versé 25 ml dans des boîtes de Pétri) et incuber une nuit à 30 ° C. </li><li> Choisir une colonie en utilisant une cheville en bois stérile et inoculer à 5 ml de liquide YPD (10 g d&#39;extrait / l de levure, 20 g / L de peptone, 20 g / l de dextrose, autoclavé et aseptique aliquoté dans 5 ml de culture en verre de 16 x 150 mm tubes). </li><li> Incuber pendant une nuit à 30 ° C dans un rouleau tambour à 60 tours par minute. </li><li> Prélever 1 ml de C. culture albicans et le transfert dans un tube à centrifuger stérile de 1,7 ml. </li><li> Centrifuger à 5000 g pendant quelques secondes. Vider le surnageant et ajouter 1 ml de PBS stérile, vortex soigneusement. Répéter deux fois. </li><li> Compter les colonies en utilisant un hémocytomètre en diluant le 1 ml stock 1: 1000 dans du PBS stérile. </li></ol> 4. Injection de poisson zèbre dansla vessie natatoire <ol><li> Réchauffer un plat d&#39;injection (2% d&#39;agarose) pendant 30 min dans un incubateur à 33 °. </li><li> Au moment désiré, retirer 100 des 150 ml de l&#39;E3 + PTU du plat 15 cm de Pétri contenant le poisson d&#39;injecter en utilisant une pipette de 25 ml, sans aspirer toute poisson zèbre. </li><li> Anesthésier 4 dpf poisson zèbre en ajoutant 2 ml d&#39;un 4 mg / ml tamponnée tricaïne méthane sulfonate stock (TR) pour les 50 ml restants médias (concentration finale de 200 pg / ml) et attendre 15 min. </li><li> Sous un microscope de dissection, choisir des poissons avec une vessie natatoire gonflée. Le poisson peut également être projeté à ce moment pour phénotype homogène, comme la distribution des neutrophiles dans mpo: ligne GFP en utilisant un microscope à dissection épifluorescence. </li><li> Diluer le C. albicans stock à partir de sa concentration d&#39;origine (étape 3.8) à 1,5 x 10 7 unités formatrices de colonies par ml (cfu / ml) dans du PBS formage. </li><li> Transférer 30 poisson zèbre sur le plat d&#39;injection à l&#39;aide d&#39;un tuyau de transfert en plastiqueTTE et retirez l&#39;excès médias. </li><li> En utilisant un outil de pêche fil (0,012 pouce la ligne de pêche de diamètre super-collés dans un capillaire de borosilicate), faire 3 lignes de 10 poissons, en tenant le plat d&#39;injection verticale et orienter le poisson verticalement. </li><li> Retirer l&#39;excès médias. </li><li> Bien agiter au vortex le tube avec 1,5 x 10 7 ufc / ml C. albicans. </li><li> Remplir une micro-aiguille avec 3 pi de C. albicans en utilisant une nouvelle pointe de pipette microloader et définir la durée d&#39;impulsion sur le réglage approprié (voir l&#39;étape 2.9). </li><li> Tourner le plat d&#39;injection pour faire un angle de 20 ° entre la micro-aiguille et la tête du poisson, visant vers l&#39;arrière de la vessie natatoire (figure 3A). </li><li> Poussez la micro-aiguille dans la vessie natatoire, assurant que le trou de l&#39;aiguille est dans la lumière, et appuyez sur la pédale fois. </li><li> Répétez l&#39;opération pour chaque poisson et de les transférer à un bac de récupération (25 ml E3 + PTU) en inondant le plat avec un E3d vider dans le bac de récupération. </li><li> Transbordement, un autre 30 poissons à l&#39;antenne d&#39;injection. </li><li> Répéter l&#39;injection avec une nouvelle micro-aiguille remplie de fond au vortex C. albicans. </li><li> Finition en injectant le contrôle PBS, si nécessaire, en utilisant une boîte d&#39;injection différent pour éviter la contamination et les transférer à un plat de récupération séparée (étape 4.10). </li></ol> 5. Dépistage du poisson pour inoculum désiré <ol><li> Préparer 40 ml de 0,4% bas point de fusion solution d&#39;agarose (LMA) en ajoutant 160 mg d&#39;agarose à bas point de fusion 40 ml de E3, l&#39;ébullition par micro-ondes jusqu&#39;à dissolution et refroidissement à 37 ° C. Ajouter 400 ul de TR (concentration finale 200 ug / ml) une fois refroidi. </li><li> Après 30 min de récupération, anesthésier le poisson comme décrit (étape 4.3 en utilisant seulement 1 ml de TR dans 25 ml E3 + PTU, concentration finale de 200 pg / ml). </li><li> Transfert 30 poissons (avec aussi peu que possible les médias) à 2 ml de LMA dans un bateau de poids. </li><li> Aspirer chaque poisson avec une pipette de transfert et plaque le poisson avec une gouttes de LMA dans une plaque d&#39;imagerie 96 puits, en utilisant seulement les 10 x 6 puits central. </li><li> Répétez jusqu&#39;à ce que tous les poissons à l&#39;écran sont chargés à la plaque d&#39;imagerie. </li><li> Que les médias se solidifient à la température ambiante pendant 5 min. </li><li> Placez le poisson de leur côté, en se assurant qu&#39;ils sont en contact avec le fond de verre. </li><li> En utilisant l&#39;objectif 20X sur une épifluorescence / microscope confocal inversé et le filtre approprié, enregistrer le nombre de C. des cellules de levure dans la vessie natatoire albicans de chaque poisson. </li><li> Sélectionnez le poisson avec de 15 à 25 cellules de levure dans la vessie natatoire (ou inoculum désiré), euthanasier non sélectionnée poissons (800 ug / ml de TR en E3) et revenir poissons sélectionné pour une boîte de Petri profonde avec 50 ml de l&#39;E3 + PTU par ajoutant 3 gouttes de E3 à la plaque de formation d&#39;image sélectionnés puits et en aspirant le poisson avec une pipette de transfert en plastique. </li><li> Incuber à 33 ° C pendant la durée souhaitée. </li></ol> 6. Imagerie du poisson post-dépistage <ol><li> Au moment désiré, retirer 25 des 50 ml de milieu E3 + PTU du plat profond Petri contenant le poisson à l&#39;image. </li><li> Anesthésier le poisson comme décrit (étape 4.3 en utilisant seulement 1 ml de TR dans 25 ml E3 + PTU reste, concentration finale de 200 pg / ml). </li><li> Plate le poisson dans une plaque d&#39;imagerie de 96 puits et la position appropriée (étapes 5.3 à 5.7). </li><li> Image par microscopie confocale en utilisant les lasers appropriés et les filtres d&#39;émission. Première accent sur le poisson en utilisant l&#39;objectif 4X vertu contraste interférentiel différentiel (DIC) et acquérir des images de la région d&#39;intérêt en utilisant l&#39;objectif 20X avec les lasers confocale en mode de balayage avec 2 vitesses ps / pixel et 1024 x 600 ratio d&#39;aspect de pixel. Acquérir Z-piles avec une taille de pas um, en appliquant le mode de filtre de Kalman de 3 images. </li><li> Retour à une boîte de Petri profonde avec 50 ml E3 + PTU ou en individual puits (plat de 24 puits de culture, 3 ml E3 + PTU) et incuber à 33 ° C. </li></ol> 7. Dissection de la vessie natatoire <ol><li> Remplir une seringue de 10 ml avec de la graisse à vide élevé. </li><li> Préparer un LMA 2% (étape 5.3 avec 160 mg de bas point de fusion agarose dans 8 ml de l&#39;E3). </li><li> À l&#39;heure souhaitée, anesthésier le poisson (étape 5.2). </li><li> 10 Transfert des poissons dans un tube de centrifugeuse de 1,7 ml avec 1 ml de E3 et euthanasie par addition de 200 ul de 4 mg / ml de tricaïne (concentration finale de 800 ug / ml) dans le tube de centrifugation et en incubant pendant 15 min à température ambiante. </li><li> Préparer une lame de verre de formation d&#39;image en plaçant quatre gouttes de graisse à vide dans un carré, 1 cm de distance sur une microlame (75 x 25 mm, figure 2). </li><li> Placez deux gouttes de 2% LMA dans le milieu de la place. </li><li> Placez un poisson sur une lame de verre séparée sous un microscope de dissection et vérifiez que le cœur a cessé de battre, retirez l&#39;excès d&#39;eau. </li><li&gt; en utilisant des pincettes fines disséquer la vessie natatoire en maintenant la tête dans une position fixe avec les pinces gauche et tirant vers le bas l&#39;intestin antérieur avec les bonnes pincettes (figure 4A). </li><li> Ramassez la vessie natatoire par le conduit pneumatique avec les pincettes gauche, le séparant du tube digestif, et le placer au centre de la LMA 2% avant qu&#39;il se solidifie. </li><li> Placez une lamelle (18 x 18 mm) sur le dessus de la lame de verre d&#39;imagerie et poussez-le jusqu&#39;à ce qu&#39;il touche la graisse à vide ainsi que le LMA (Figure 2). </li><li> Image à l&#39;intérieur 10 min de la dissection comme décrit dans l&#39;étape 6.4. Répétez l&#39;étape 7.7 à 7.11 avec les autres poissons. </li></ol>

The authors have nothing to disclose.

Les progrès et les limites du modèle de la maladie de la micro-injection de vessie natatoire Le modèle présenté ici est une extension du modèle d&#39;immersion de la candidose des muqueuses décrit dans Gratacap et al (2013).; il ajoute les avantages d&#39;un temps d&#39;infection contrôlée, une dose d&#39;infection hautement reproductibles, et donc une meilleure efficacité. Nous démontrons ici de nouvelles méthodes qui permettent la documentation temporelle non-invasive de ...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name</td>
			<td style="width:259px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL tubes</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deep Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR Scientific</td>
			<td>90000-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>VWR Scientific</td>
			<td>90000-264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>VWR Scientific</td>
			<td>90000-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine tweezers (Dumont Dumoxel #5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wooden Dowels</td>
			<td>VWR Scientific</td>
			<td>10805-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melt Agarose</td>
			<td>VWR Scientific</td>
			<td>12001-722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillary</td>
			<td>Sutter Instruments</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPPI-3 Injection system</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Pressure Unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foot Switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scope</td>
			<td>Olympus</td>
			<td colspan="2">SZ61 top SZX-ILLB2-100 base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x microscope objective</td>
			<td>Olympus</td>
			<td>UPlanSApo 20x/0.75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller drum</td>
			<td>New Brunswick Scientific</td>
			<td>TC-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass culture tubes (16 x 150 mm)</td>
			<td>VWR Scientific</td>
			<td>60825-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>VWR Scientific</td>
			<td>BDH4534-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR Scientific</td>
			<td>BDH4532-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>VWR Scientific</td>
			<td>BDH0246-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (Corning)</td>
			<td>VWR Scientific</td>
			<td>BDH4520-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Children clay (Play-Doh)</td>
			<td>Hasbro</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>VWR Scientific</td>
			<td>VW6276-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer (Hausser scientific)</td>
			<td>VWR Scientific</td>
			<td>15170-172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type A immersion oil</td>
			<td>Blue Marble Products</td>
			<td>51935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie</td>
			<td>VWR Scientific</td>
			<td>14216-184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (Lonza)</td>
			<td>VWR Scientific</td>
			<td>12001-870</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fishing wire</td>
			<td>Stren</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well imaging plate (Sensoplate)</td>
			<td>Greiner Bio-One</td>
			<td>655892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease (Dow Corning)</td>
			<td>VWR Scientific</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microslide (25 x 75 mm)</td>
			<td>VWR Scientific</td>
			<td>48300-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips (18 x 18 mm), No 1.5</td>
			<td>VWR Scientific</td>
			<td>48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm Petri dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>32-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol (EMD chemicals)</td>
			<td>VWR Scientific</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well culture dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight boats (8.9 cm)</td>
			<td>VWR Scientific</td>
			<td>89106-766</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

La défense précoce contre les agents pathogènes des muqueuses consiste à la fois une barrière épithéliale et les cellules immunitaires innées. Le immunocompétence des deux, et de leur intercommunication, sont primordiaux pour la protection contre les infections. Les interactions de cellules immunitaires innées et épithéliales avec un agent pathogène sont les mieux étudiés in vivo, où le comportement complexe se déroule dans le temps et l&#39;espace. Cependant, les modèles existants ne permettent pas facile imagerie spatio-temporelle de la bataille avec des agents pathogènes au niveau de la muqueuse. Le modèle développé ici crée une infection de la muqueuse par injection directe de l&#39;agent pathogène fongique, Candida albicans, dans la vessie natatoire du poisson zèbre juvénile. L&#39;infection résultant permet imagerie à haute résolution du comportement des cellules épithéliales et immunitaire innée à travers le développement de la maladie des muqueuses. La polyvalence de cette méthode permet pour interrogatoire de l&#39;hôte à sonder la séquence détaillée des événements immunitaire conduisant à phagocyte recrutement et d&#39;examiner les rôles de types cellulaires particuliers et voies moléculaires dans la protection. De plus, le comportement de l&#39;agent pathogène en fonction de l&#39;attaque immunitaire peut imager simultanément en utilisant la protéine fluorescente exprimant C. albicans. Résolution spatiale accrue de l&#39;interaction hôte-pathogène est également possible en utilisant la technique de dissection vessie natatoire rapide décrite. Le modèle d&#39;infection de la muqueuse décrite ici est simple et hautement reproductible, ce qui en fait un outil précieux pour l&#39;étude de la candidose buccale. Ce système peut également être largement traduisible à d&#39;autres pathogènes des muqueuses comme les microbes mycobactériennes, bactériennes ou virales qui infectent normalement à travers des surfaces épithéliales.

La micro-injection dans la vessie natatoire postérieure La méthode expérimentale présentée ici décrit l&#39;injection d&#39;une dose constante de C. des cellules de levure albicans dans la vessie natatoire des 4 dpf poisson zèbre. Des travaux antérieurs avec le modèle d&#39;immersion suggère que la réponse immunitaire de la vessie natatoire à C. albicans est similaire à mammifère candidose des muqueuses 32. Ici, nous démontrons une méthode d...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modélisation des muqueuses candidose larves de poisson zèbre par injection vessie natatoire

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K vues.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Vidéo: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

L&#39;imagerie non invasive de la candidose disséminée chez les larves poisson zèbre

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Recherche

Immunologie et infection

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Essai de coculture pour évaluer la migration transépithéliale des neutrophiles induite par l’agent pathogène

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagerie non invasive de la réponse immunitaire innée dans un modèle de poisson zèbre larves de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Échantillonnage non invasive de muqueuse liquide pour la Quantification des niveaux d’immunitaire-médiateur In Vivo des voies respiratoires supérieures

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

La Formation de Biofilm induite sur les sels biliaires chez les agents pathogènes entériques : Techniques d’Identification et de Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modélisation de la tuberculose chez Mycobacterium marinum adultes infectés poisson zèbre

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Le modèle d’Infection de teigne Galleria mellonella pour candidose disséminée

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Induction de blessure pulmonaire de souris par injection endotrachéale de bleomycine

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Cibler les médicaments aux macrophages de poisson zèbre larvaire en injectant des liposomes chargés de drogues

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Isolement des cellules monnucléaires tonsillar pour étudier les réponses immunitaires innées Ex Vivo dans un tissu lymphoïde muqueuse humain

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...